Coffee contains a highly complex mixture of flavour molecules. Extensive research on the composition of instant and fresh ground coffee beverages has, to date, identified more than 850 compounds, many of which are flavour active molecules (Flament, I (2002) Coffee Flavor Chemistry, John Wiley and Sons, UK). However, few of the final coffee flavour molecules found in the cup of coffee are present in the raw material, the green grain (green beans) of the plant species Coffea arabica or Coffea canephora (robusta). In fact, the majority of the coffee flavour compounds are generated during one or more of the multiple processing steps that occur from the harvest of the ripe red coffee cherries to the final roasted ground coffee product, or extracts thereof, for example soluble coffee products.
The various steps in the production of coffee are described in Smith, A. W., in Coffee; Volume 1: Chemistry pp 1-41, Clark, R. J. and Macrea, R. eds, Elsevier Applied Science London and New York, 1985; Clarke, R. J., in Coffee: Botany, Biochemistry, and Production of Beans and Beverage, pp 230-250 and pp 375-393; and Clifford, M. N. and Willson, K. C. eds, Croom Helm Ltd, London. Briefly, the process starts with the collection of mature, ripe red cherries. The outer layer, or pericarp, can then be removed using either the dry or wet process. The dry process is the simplest and involves 1) classification and washing of the cherries, 2) drying the cherries after grading (either air drying or mechanical drying), and 3) dehusking the dried cherries to remove the dried pericarp. The wet process is slightly more complicated, and generally leads to the production of higher quality green beans. The wet process is more often associated with C. arabica cherries. The wet process consists of 1) classification of the cherries, 2) pulping of the cherries, this step is done soon after harvest and generally involves mechanical removal of the “pulp”, or pericarp, of the mature cherries, 3) “fermentation”, the mucilage that remains attached to the grain of the cherries after pulping is removed by allowing the grain plus attached mucilage to be incubated with water in tanks using a batch process. The “fermentation” process is allowed to continue up to 80 hours, although often 24 hours is generally enough to allow an acceptable fermentation and to cause the pH to drop from around 6.8-6.9 to 4.2-4.6, due to various enzymatic activities and the metabolic action of microorganisms which grow during the fermentation, 4) drying, this step involves either air or mechanical hot air drying of the fermented coffee grain and 5) “hulling”, this step involves the mechanical removal of the “parch” of the dried coffee grain (dried parchment coffee) and often the silverskin is also removed at this stage. After wet or dry processing, the resulting green coffee grain are often sorted, with most sorting procedures being based on grain size and/or shape.
The next step in coffee processing is the roasting of the green grain after dehusking or dehulling of dry or wet processed coffee, respectively. This is a time-dependent process which induces significant chemical changes in the bean. The first phase of roasting occurs when the supplied heat drives out the remaining water in the grain. When the bulk of the water is gone, roasting proper starts as the temperature rises towards 190-200° C. The degree of roasting, which is usually monitored by the colour development of the beans, plays a major role in determining the flavour characteristics of the final beverage product. Thus, the time and temperature of the roasting are tightly controlled in order to achieve the desired coffee flavour profile. After roasting, the coffee is ground to facilitate extraction during the production of the coffee beverage or coffee extracts (the latter to be used to produce instant coffee products). Again, the type of grinding can influence the final flavour of the beverage.
While a considerable amount of research has been carried out on the identification of the flavour molecules in coffee, much less work has been done regarding the physical and chemical reactions which occur within the coffee grains during each of the processing steps. This latter point is particularly evident for the roasting reaction, where the large number of grain constituents undergo an extremely complex series of heat induced reactions (Homma, S. 2001, In “Coffee: Recent Developments”. R. J. Clarke and O. G. Vitzthum eds, Blackwell Science, London; Yeretzian, C., et al ((2002) Eur. Food Res. Technol. 214, 92-104; Flament, I (2002) Coffee Flavor Chemistry, John Wiley and Sons, UK; Reineccius, G. A., “The Maillard Reaction and Coffee Flavor” Conference Proceedings of ASIC, 16th Colloque, Kyoto, Japan 1995).
While the details of most of the reactions that occur during the different steps of coffee processing remain relatively unclear, it is thought that an important flavour generating reaction responsible for many of the flavours associated with coffee aroma is the “Maillard” reaction during coffee roasting. A vigorous Maillard reaction occurs between the grain reducing sugars/polysaccharide degradation products and the amino group containing molecules (particularly the proteins, peptides, and amino acids) during the roasting step.
Because the Maillard reaction apparently makes an important contribution to the generation of coffee flavour and aroma molecules during coffee roasting, there might be an association between the levels of primary Maillard reactants in the green beans and the quality of the flavour/aroma developed after roasting.
As noted above, an important group of substrates in the Maillard reaction are amino acids, peptides and proteins. Using 2-D electrophoresis, it has been shown that differences exist in the levels and amounts of the major storage proteins in arabica and robusta green coffee beans—however, no association between these storage protein differences and flavour quality was noted (Rogers et al, 1999, Plant Physiol. Biochem. Vol 37, 261-272). It has also recently been found that small differences exist between the storage proteins of immature and mature coffee beans, which have different flavour qualities (Montavon, P. et al, 2003, J. Agric and Food Chemistry Vol 51, 2328-2334). Because there are many changes occurring during seed maturation, this latter work suggests a link may exist between the quality improvement caused by maturation and the differences seen in the 2-D gel patterns of the main coffee storage proteins.
It has recently been shown that there are differences in the profiles of peptides isolated from arabica and robusta green beans (Ludwig et al 2000, Eur. Food Res Technol., Vol 211, 111-116.). Although their results showed that the arabica and robusta peptide extracts differ in their aroma precursor profile, the data presented in this report do not identify which component(s) in the extracts is/are responsible for these aroma profile differences. These workers also detected at least two different proteinase activities in crude extracts of the green coffee, but they did not correlate any specific activities with aroma/flavour quality (Ludwig et al 2000, Eur. Food Res Technol., Vol 211, 111-116). Finally, it is also thought that the very high temperatures used during the later stages of green coffee grain roasting cause substantial cleavage of the proteins present in the coffee grain (Homma, S. 2001, In “Coffee: Recent Developments”. R. J. Clarke and O. G. Vitzthum eds, Blackwell Science, London; Montavon, P., et al 2003, “Changes in green coffee protein profiles during roasting”, J. Agric. Food Chem. 51, 2335-2343). However, the overall scheme for this protein degradation is very poorly understood, but presumably depends on, among other things, the precise state of the main coffee proteins in the raw material before the start of roasting. To our knowledge, there are no other significant reports addressing the possibility that peptide profiles in coffee could be involved in the production of coffee aroma/flavour.
In the roasting of the fermented seeds of Theobroma cacao (cocoa beans), there would appear to be an involvement of seed amino acids and peptides in the development of Maillard reaction aromas/flavours. Relative to other seeds, T. cacao seeds have been shown to have an unusually high level of aspartic proteinase activity (Biehl, B., Voigt, J., Voigt, G., Heinrichs, H., Senyuk, V. and Bytof, G. (1994) “pH dependent enzymatic formation of oligopeptides and amino acids, the aroma precursors in raw cocoa beans”. In The Proceedings of the 11th International Cocoa Research Conference, 18-24 Jul. 1993, Yamoussoukro, Ivory Coast). In order to produce cocoa beans with a high level of cocoa flavour precursors, it is necessary to carry out a natural fermentation step (unfermented beans develop little flavour when roasted). During this fermentation step, the sugars in the pulp are fermented, generating high levels of acids, particularly acetic acid (Carr, J. G. (1982) Cocoa. In Fermented Foods. Economic Microbiology. Vol 7. pages 275-292. (A. H. Rose ed). Academic Press). As the fermentation continues, the pH in the seed decreases and the cell structure becomes disrupted. The low pH triggers the abundant cacao seed aspartic proteinase to become mobilized and/or activated, resulting in a massive degradation of cellular protein (Biehl, B., Passern, D., and Sagemann, W. (1982) “Effect of Acetic Acid on Subcellular Structures of Cocoa Bean Cotylydons”. J. Sci. Food Agric. 33, 1101-1109; Biehl, B., Brunner, E., Passern, D., Quesnel, V. C., and Adomako, D. (1985) “Acidification, proteolysis and flavour potential in fermenting cocoa beans”. J. Sci. Food Agric. 36, 583-598). Peptides and amino acids have been shown to be cocoa flavour precursors (Rohan, T. (1964) “The precursors of chocolate aroma: a comparative study of fermented and unfermented cocoa beans”. J. Food Sci., 29, 456-459; Voigt, J. and Biehl, B. (1995) “Precursors of the cocoa specific aroma components are derived from the vicilin-class (7S) globulin of the cocoa seeds by proteolytic processing”. Bot. Acta 108, 283-289). Thus, the T. cacao seed asp artic proteinase, together with a seed serine carboxypeptidase, have been proposed to be critical for the generation of cocoa flavour precursors during fermentation (Voigt, J. and Biehl, B. (1995) “Precursors of the cocoa specific aroma components are derived from the vicilin-class (7S) globulin of the cocoa seeds by proteolytic processing”. Bot. Acta 108, 283-289; Voigt, J., Heinrichs, H., Voigt, G. and Biehl, B. (1994) “Cocoa-specific aroma precursors are generated by proteolytic digestion of the vicilin-like globulin of cocoa seeds”. Food Chemistry, 50, 177-184.) The gene encoding the abundant cacao seed aspartic proteinase has been identified and a method to over-express this protein in cacao seeds which can generate increased levels of cacao flavour precursor amino acids and peptides in fermented cocoa beans has recently been described in International Patent Publication No. 02/04617, the whole contents of which are incorporated herein by reference. However, the teaching of International Patent Publication No. 02/04617 is directed towards cacao seeds, which undergo a specific long acid fermentation step, unlike coffee grains which do not.
An important vacuolar cysteine proteinase (CP) is the KDEL (SEQ ID NO: 17) containing cysteine proteinase. This type of proteinase has been characterized in several plants. To date, three genes encoding cysteine proteinases with C-terminal KDEL (SEQ ID NO: 17) sequences have been found in arabidopsis (Gietl, C., and Schmid, M. 2001, Naturwissenschaften 88, 49-58). One is expressed in senescing ovules, one in vascular vessels, and the third in maturing siliques. However, more detailed studies on this protein have been done in other plants. For example, a CP called the sulfhydryl-endoproteinase (SH-EP) has been characterized in the cotyledons of Vigna mungo seeds (Toyooka, K., Okamoto, T., and Minamikawa, T. (200) J. Cell Biol. 148, 453-463.). SH-EP is expressed de-novo in germinating cotyledons of V. mungo, and is proposed to be involved in the degradation of storage proteins accumulated in the protein storage vacuoles (Okamato, T. and Minamikawa, T. J. Plant Physiol. 152, 675-682). A key feature of the SH-EP polypeptide is that it possesses a specific COOH terminal sequence KDEL (SEQ ID NO: 17) which directs the transport of this protein from the endoplasmic reticulum (ER) to the protein storage vacuoles (Toyooka et al., 2000). It has also been recently proposed that the SH-EP protein is actually involved, via the presence of its KDEL (SEQ ID NO: 17) sequence, in the formation of specific vesicles called KV (KDEL Vesicles) in a previously undescribed vesicle transport system (Okamato, T., Shimada, T., Hara-Nishimura, I., Nishimura, M., and Minamikawa, T. (2003) Plant Physiology, 132, 1892-1900).
A related proposal has been made for a KDEL (SEQ ID NO: 17) containing CP protein found in germinating castor bean cotyledons (Ricinus communis). In this plant, the authors implicate this KDEL (SEQ ID NO: 17) proteinase in the programmed cell death of the endosperm to continue suppling nutrients for the germinating castor bean embryo (Gietl, C., and Schmid, M. 2001, Naturwissenschaften 88, 49-58). These authors propose that, in the castor bean, the KDEL (SEQ ID NO: 17) proteinase is made in the ER of germinating seeds before day 3. When the seed coat is cast off, around day 3, the KDEL (SEQ ID NO: 17) containing CP then gets packaged into a specific vesicle called a ricinosome. Later, as the endosperm becomes soft between day 4-5, the KDEL (SEQ ID NO: 17)—CP has its anchor sequence (KDEL) (SEQ ID NO: 17) cleaved off and this proteinase migrates to the cytoplasm where it assists in the general degradation of the cellular protein.